Thymoquinone: Flow Therapeutic Analysis

1. Introduction

Thymoquinone (TQ) have been mainly considered for its anticancer activity. TQ’s first isolated from a natural product of the Nigella sativa L. black seed, one of the most used herb in folk medicine in the Mediterranean area and West Asia. Nigella sativa is well-known for its healing potential (Gali-Muhtasib et al., 2006). Scientific research supports the country people medicine utilization of the TQ as an anti inflammatory, antimicrobial and anticancer agent both in vitro and in vivo (Gali-Muhtasib et al., 2006).There is powerful evidence that TQ induce apoptosis in many cancer cell lines by different pathways (Arafa et al., 2011; Badr et al., 2011; Dergarabetian et al., 2013; Martin et al., 2006; Ng et al., 2011; Schneider-Stock

The main problems are auto flourescence (AF) and the selection of the most suitable fluorescent dyes that had been originally developed for mammalian cells (Steen, 2000; Tracy et al., 2010; Vives-Rego, 2000)

Flow cytometric analysis of mammalian cells is usually complicated by high proportions of AF. The AF range of most cell types is typically very vast; the emission ranges from 500 to 700 nm, with a peak emission at 550 nm (Vives-Rego, 2000). Thus the background caused by AF is a typical problem with fluorescein-conjugated probes (Roederer and Murphy, 1986). These possibility discrepancies point to the need for careful protocol development and choice of proper controls to extract reliable and biologically relevant information from flow cytometry assay (Renggli et al., 2013; Tracy et al., 2010)

In the present study, it was attempted to overcome some of these problem. It became apparent that TQ increase AF under standard conditions in flow cytometric analysis. These background signals need to be taken into account when cell lines are evaluated with fluorescent

Annexin V-FITC was used as a marker of phosphatidylserine exposure and PI as a marker for dead cells. This combination allows differentiation among early apoptotic cells (annexinV-positive, PI-negative), late apoptotic/necrotic cells (annexin V-positive, PI-positive), and viable cells (annexin V-negative, PI-negative). Cells were seeded and treated with the indicated compounds or solvent for the cell apoptosis. After 48 h of treatment, the untreated and treated cells were harvested and washed with cold PBS, and aliquots of 5×105 cells were washed with PBS and resuspended in 500 μl of binding buffer 1X provided with the kit. A volume of 5 μl of Annexin V-FITC and 5 μl of PI were added to 100 μl of cells suspension and cells were incubated at room temperature in the dark for 30 min. Approximately 20,000 cells were analyzed by flow cytometry using a flow cytometer (partec, Milton Keynes, UK) with a 488-nm blue laser. The percentage of cells in each category was determined. The experiments were performed twice and gave similar